EMBL-EBI
MSD database is
structured around the fact
that Proteins are “sticky”
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A short biography of 1 protein
whose very existence depends
on being as sticky as possible
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EMBL K02078
ttaacgcgta
ccaataaaaa
ctacttccgc
ataaacgcat
gcagttgtcg
tgaggctttc
cttatcgagc
gcctaccaag
caaaaatcag
tctgccggcg
gttaaaaacg
ggcaaaaaac
cagccggtta
gacaccaagc
ttaggcctta
ccgggcggct
taatatatcg
aattcaaaaa
agtaacgaaa
aaaccacacc
aaaatttcac
caacaaaaaa
ccctttcaat
tgatgattgt
actacaccgc
ccgtcaccga
tggcatcccc
gcgtcgttac
tctccctgtg
cgcgcaccga
acctgccgtc
aattttaaat
tgtcttttaa
at
tctcaaattc
atcggcacta
cacctaaaag
ctcaaaacat
ccgatggtta
taggagtaat
gatcgctatc
ccgcgcgcaa
gtattacctg
cccctccgac
cgccacaatg
ggccaggcgt
cgacgacacc
aacctgccgc
aaatcaagcg
gggtttgcaa
cgacccaatc
aaactgacaa
aaaatacaaa
aaaatcggca
aatacattgc
tttatgaata
gtcggcattt
gtttccgaag
aatcacggca
atcaaaggca
ctttcaagcg
gaaaacggtt
gttgccgacg
gataaggcat
gtaagtgatt
ggcgggcggg
aacacacccg
ttttcgacac
ataaaaacaa
cgaatcttgc
atgatgccga
cccttcaaaa
tggcggcagt
ccatcctttt
aatggccgga
aatatgttaa
gcgtaaacaa
cggtaaaatg
ccaaagacgg
ctgatgccaa
ttccacccgc
gtcgtccgtt
ataccccatg
tgccgccccc
ttatatagag
tttataatac
tggcaagccc
aggctttacc
cgcccttccc
ggccgaaggt
aaacaacact
agaggttgaa
tgaaatcaaa
gttctgcgga
caaagaaatc
atgaggcaaa
ccggatcaac
ccggtggaaa
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UniProt P02974
MNTLQKGFTL
YQDYTARAQV
HGKWPENNTS
KNGVVTATML
NGSVKWFCGQ
TKHLPSTCRD
IELMIVIAIV
SEAILLAEGQ
AGVASPPSDI
SSGVNNEIKG
PVTRTDDDTV
NFDAK
GILAAVALPA
KSAVTEYYLN
KGKYVKEVEV
KKLSLWARRE
ADAKDGKEID
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PDB 1AY2
MSD DATABASE
pentamer
MSD DATABASE
negatively stained TEM images
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The pili are polar flexible filaments of about 5.4 nm
diameter and 2500 nm average length.
Neisseria gonorrhoeae expressing pili
and interacting with epithelial cells.
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PDB 2PIL / 1AY2
Forest, K.T., Parge, H.E., Tainer, J.A. Nature. 1995 378, 32-8.
Forest, K.T., Dunham, S.A., Koomey, M., Tainer, J.A. Mol Microbiol.
1999 31, 743-52.
SOURCE: Neisseria gonorrhoeae
A fibre forming cell adhesion protein responsible for the
virulent attachment
Pilin is a subunit of the pilus, a polar flexible filament, which
consists of a single polypeptide chain arranged in a helical
configuration of five subunits per turn.
PDB 1ay2 , 1dzo , 1hpw , 1kb7 , 1nil , 1pan
CATH 3.30.700.10
SCOP d.24.1.1 , j.23.1.1
Type IV Pilin Structure and
Assembly: X-Ray and EM
Analyses of Vibrio cholerae
Toxin-Coregulated Pilus and
Pseudomonas aeruginosa PAK
Pilin
L. Craig, R.K. Taylor, M.E.
Pique, B.D. Adair, A.S. Arvai, M.
Singh, S.J. Lloyd, D.S. Shin,
E.D. Getzoff, M. Yeager, K.T.
Forest & J.A. Tainer
Molecular Cell, 11, 1139–1150,
2003
EM Analysis of TCP Reveals a Three-Start Helix with a 45 Pitch
C Bundle of negatively stained TCP filaments
D computed Fourier transform of a single filament within the bundle
as indicated by the box in (C).
E Left-handed representation of a three-start helix with each start
shown in a different colour.
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Pili are thin, protein tubes
The pilus has a shaft composed pilin.
At the end of the shaft is the adhesive tip structure
having a shape corresponding to that of specific
glycoprotein or glycolipid receptors on a host cell
 Because both the bacteria and the host cells have a
negative charge, pili may enable the bacteria to bind
to host cells without initially having to get close
enough to be pushed away by electrostatic
repulsion.
Once attached to the host cell, the pili can
depolymerize and enable adhesions in the bacterial
cell wall to make more intimate contact.
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Type IV pili are not merely passive sticky fibres
but dynamic machines that participate in a
surprising number of functions including:
Bacterial aggregation
Adhesion to host cells
Twitching motility
Pilus retraction
DNA transformation
In another bacterial species, motility.
Phage receptor in V. cholerae.
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EMBL
UniProt
PDB
Assembly (MSD)
Microscopy
still not the full story - GENOME
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Pilus gene organisation
 Many copies of pilin gene throughout
chromosome
 Two are functional, pilE1 and pilE2
 All other copies are silent
 Antigenic variation occurs due to
recombination (within mini-cassettes)
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Antigenic variation in N. gonorrhoeae
 A single cell can give rise to daughter cells
expressing structurally and antigenically
different pili
 Gonococcus has the genetic capacity to make
as many as a million different pilin variants
 All able to bind to same host tissues and to
cause the same disease symptoms
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PILI are the pathogen’s answer to
Mankind's physical defence systems
One of the body's innate defences is the
ability to physically remove bacteria from the
body by:
 constant shedding of surface epithelial
cells
 coughing, sneezing, vomiting, and
diarrhoea
 removal by bodily fluids such as saliva,
blood, mucous, and urine.
pili enable adhere N. gonorrhoeae to
receptors on target epithelial cells and thus
colonize and resist flushing by the body.
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REMEMBER – this all
achieved by simple
non-covalent forces
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What has all this got to do with MSD?
PDB Entries and X-Ray results
1. Crystal Structure
2. Molecular Structure (covalent)
3. Oligomeric Assembly
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MSD Relational Database
Exp. Result
Assembly
Chains
Residues
assembly_deposition_fk
ASSEMBLY
assembly_deposition_fk
CHAIN
chain_assembly_fk
DEPOSITION
chain_assembly_fk
assembly_a_data_fk
atd_chain_fk
atd_chain_fk
COMPONENT
ATOM DATA
atd_component_fk
atd_component_fk
assembly_a_data_fk
ASSEMBLY DATA
atd_model_fk
atd_atom_fk
ATOM
atd_atom_fk
assembly_data_model_fk
assembly_data_model_fk
atd_model_fk
atd_alt_fk
MODEL
ALT
atd_alt_fk
Atoms
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KEY to MSD DataBase
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Biological Context
PDB  MSD
Oxalate oxidase 1FI2 hexameric
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Protein Stickiness
What does this mean?
What is the evidence?
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PDB Xray coordinates
 PDB entry the deposited coordinates
usually consist of the contents of the
asymmetric unit:
 The contents of the ASU define a single copy of the
macromolecule
 The contents of the ASU consist of more than one copy of the
macromolecule
 The contents of the ASU require crystallographic symmetry
operations to be applied to generate the complete
macromolecule(s)
 A combination of the above, including multiple copies and
required symmetry transformations
A crystal is a periodic arrangement of a motif in a lattice. The
motif can be a single atom, a small molecule, a protein or any
combination thereof. Often the motif, also referred to as to the
'asymmetric unit', is subjected to a number of symmetry
operations yielding differently oriented copies.
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Space Groups
The combination of all available symmetry
operations (point groups plus glides and screws)
with the Bravais translations leads to exactly 230
combinations, the 230 Space Groups.
Kathleen Yardley Lonsdale 19031971. Carried out a profound and
systematic study of the theory of
space groups , methods for their
determination, and the possibilities
of molecular symmetry that are
involved (1924, 1936).
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benzene
C6H6
Covalent bonded
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Benzene crystallised in
Space Group P6/m
6-fold rotation axis
Mirror plane
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Benzene P6/m in the PDB
Entire atomic contents:
ATOM C1 x1 y1 z1 occupancy 0.5
ATOM H1 x2 y2 z2 occupancy 0.5
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HELD TOGETHER BY WEAK FORCES
The stronger of the two is the hydrogen bond.
The weaker is the van der Waal's forces.
Both interactions depend on the same
fundamental cause, the charge on electrons,
and how that results in attraction and repulsion
at an atomic level.
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Johannes D. van der Waals
The equation of state for
gases and liquids
Nobel Prize 1910
The origin of the London van der Waals
force lies in the instantaneous dipole
generated by the fluctuation of electron
cloud surrounding the nucleus of
electrically neutral atoms.
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van der Waals forces
All intermolecular attractions are known
collectively as van der Waals forces. The various
different types were first explained by different
people at different times.
Dispersion forces, for example, were described
by London in 1930;
dipole-dipole interactions by Keesom in 1912.
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van der Waals
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Hydrogen Bonding
Linus Pauling (1901-1994)
Nobel Prize 1954
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Hydrogen Bonds
Pauling in 1935 was the first to explain the mysterious
stickiness of water molecules.
The basic principle behind hydrogen bonding is that
the electron deficient hydrogen atom of one polar
molecule is attracted to the electron rich side of
another polar molecule.
Hydrogen bonds are somewhat stronger than van der
Waal's forces, and require two components:
 a donor group and
 an acceptor group.
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Hydrogen Bonds
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Quaternary Structure
 Quaternary Structure is defined as that level of
form in which units of tertiary structure
aggregate to form homo- or hetero-multimers.
 Consideration of the presence of a quaternary
state is important in the understanding of a
protein's biological function.
Crystal Structure
Crystal Structure
Oligomeric Assembly
Proteins EMBL-EBI
don’t do this –
pack by translationals
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Symmetry
There are three main types of symmetry:
 symmetry with respect to a plane (mirrors)
 symmetry with respect to a line (rotations)
 symmetry with respect to a point (inversions)
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Symmetry
 symmetry with respect to a line (rotations)
 symmetry with respect to a plane (mirrors)
 symmetry with respect to a point (inversions)
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Rotational symmetry
1, 2, 3, 4, 6 -fold rotational symmetry
These are the only rotational symmetries that can exist
in crystals; all others are disallowed. These five
rotational axes are called the five Proper Axes
Symmetries showing 5-, 7-, 8-, 9-, 10-, 11-, & 13- fold
rotations are known for biological molecules – these are
observed in the Asymmetric Unit.
1g8h
A’
A
Residues of Chain A in interface
Applying 1st 3-fold Rotation
A’
Residues of Chain A’ in interface
A
A’
A”
A
Applying 2nd 3-fold Rotation
Also has a
2-fold
rotation
Final Assembly is a Hexamer from 23 symmetry
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Screw Axes
If you add translations to rotation axes, you form
what are call screw axes. For an nm screw axis,
the rotational component is 360/n degrees, and
the translations is m/n of the unit translation
along the axis.
In Biological Crystallography --> Polymers
Helices are improper Screw axes – e.g. DNA
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Screw Axes
YopM is a strongly acidic protein containing 13–20
repeats of a 19-residue leucine-rich-repeated motif
(LRR). YopM has a crescent shape, formed from parallel
β-sheets,with a loose amino terminus95.
Four YopM monomers form a hollow cylinder with an
inner diameter of 35 Å .
YopM is an important virulence factor in Yersinia
infection
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1jl5 YOPM
4-fold screw axis
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Also has a 2-fold rotation – infinite cylinder in crystal
Screw Axis
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Screw Axes example
tubulins
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Bacteriophage T4
Molecular Machine
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Bacteriophage T4
Identified
Gene products
Bacteriophage T4
1N7Z
Gp8 Baseplate Structural Protein
1QEX
Gp9
1EL6
Gp11 Baseplate-tail tube complex
1CZD
Gp45 Processivity Clamp
1G31
Gp31 Co-Chaperonin
1OCY
Receptor-Binding Domain
1RFO
Phagehead Fibritin - whisker antigen control
1C1K
Gp59 Helicase Assembly
Connector Protein From
Bacteriophage Phi29
1FOU
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Bacteriophage T4
24 Genes give proteins in the Head+Whiskers/Neck
22 Genes give proteins for the Tail+Base Plate
7 Genes give proteins in the Tail Fibres
1 Gene gives the fin attachment protein
e.g. in the Head scaffold there are 576 copies of gp22
ALL HELD TOGETHER BY WEAK FORCES
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Bacteriophage T4
Identify genes
Identify structures
Identify location
Becomes Mechanics –
just balls and springs
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Bacteriophage T4
Important
Hinge Proteins
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Hinge-Bending, Swiveling Motions
Most large proteins are built from assemblies of domains
that for the most part consist of regions of nearly rigid
motions jointed by flexible regions. The activity of many
proteins induces conformational transitions by hinge
bending, which involves the movement of relatively rigid
parts of a protein about flexible joints
The conformational switch from open to closed of the
flexible loop-6 of triosephosphate isomerase (TIM)
The hinge region on the Fc fragment of human
immunoglobulin G
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Hinge-Bending, Swiveling Motions
Hinge mechanism that occurs when there is no
continuously maintained interface constraining the
motion. Hinge motions usually occur in proteins with
two domains with one domain rotating about the
hinge as a rigid body. The rotation is caused by a few
large torsion angle changes within the hinge region.
shear mechanism that occurs when two interfaces
slide across each other in order to maintain a wellpacked interface. Shear motions are typically small
so a large shear motion will be composed of a
number of individual shear motions.
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Genes  Proteins  Structure  Function
Genes  Proteins  Structure  Function
Some Proteins have No
predicted structure or
regions predicted not to
fold
Genes  Proteins  Structure  DYNAMICS
Function
http://www.BioSimGrid.org/
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http://bioportal.weizmann.ac.il/fldbin/findex
FoldIndex© tries to answer to the question: Will
this protein fold?
It's a dynamic and interactive process that
estimates the local and general probability for the
provided sequence, under specified conditions, to
fold.
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Bacteriophage T4
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Bacteriophage T4 baseplate
Kostyuchenko et al.,
Nat. Struct. Biol.,2003
10, 688
Emd Entry 1048
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gp5/gp27 hexamer
of the tube
makes up the
hypodermic
needle at the tip
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gp5/gp27 hexamer
Bacteriophage T4
PDB Entry 1K28
BACTERIOPHAGE T4
CELL-PUNCTURING DEVICE
KANAMARU, et.al.,
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gp5/gp27 hexamer
PDB: ASU
3-fold Rotation
MSD: Assembly
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PDB Example
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Observed Asymmetric Unit
1e94 M.Bochtler
et al,
Nature, 403, 800
(2000)
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1e94 - 3 separate molecules (?)
2 dodecamers
1 hexamer
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1e94 M.Bochtler
et al,
Nature, 403, 800
(2000)
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hexamer
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hexamer
Grapple
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1e94 M.Bochtler
et al,
Nature, 403, 800
(2000)
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dodecamer
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dodecamer
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Heat shock proteins HslV and HslU that form a
new ATP-dependent protease in Escherichia coli
- ATP-dependent protease complexes rid cells of
misfolded or damaged proteins and control the
level of certain regulatory proteins.
M. Bochtler, C. Hartmann, H.K.Song, G.P.Bourenkov,
H.D.Bartunik, and R.Huber (2000) The structures of HSLU and
the ATP-dependent protease HSLU-HSLV. Nature 403, 800
Couvreur B. , Wattiez R. , Bollen A. , Falmagne P. , Le ray D. ,
Dujardin J.C. (2002). Eubacterial HslV and HslU subunits
homologs in primordial eukaryotes.Mol. Biol. Evol. 19, 21102117
Easter Island
Complex with
topknot
P31059
HSLV (P31059) caps
HSLU (P32168)
P32168
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STATS on PDB Oligomers ~ 24,000 entries
Oligomer type
Num
Monomer
7490
dimer
9179(8169)
7215(6261)
1958(1908)
trimer
1659(1628)
815( 786)
844( 842)
tetramer
2963(2930)
1852(1828)
1111(1102)
pentamer
178( 178)
hexamer
898( 883)
heptamer
octamer
53(
Homo
52)
421( 418)
91(
Hetero
91)
534( 521)
29(
29)
209( 208)
87(
87)
364( 362)
24(
23)
212( 210)
nonamer
62(
62)
6(
6)
56(
56)
decamer
72(
72)
43(
43)
29(
29)
undecamer
10(
10)
8(
8)
2(
2)
230( 230)
96(
96)
dodecamer
134( 134)
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Windscreen bug splat examples
1fmd
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SYMMETRY Rules –BUT What about -
What happened to symmetry?
2:1 hetero-complex
The Ribosome – the champion Heterocomplex
proteins tossed around the RNA
protein aggregates complicate the lives of
people who study proteins in vitro
Protein Aggregation and Amyloid Diseases
- Converting the protein from a soluble to a fibrillar
structure
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ACKNOWLEDGEMENTS
I have taken from the WWW most of the
pictures used here
The list of sources is available separately.